CHARACTERIZATION OF BKDR-DNA BINDING IN THE EXPRESSION OF THE BKD OPERON OF PSEUDOMONAS-PUTIDA

The bkd operon of Pseudomonas putida consists of the structural genes encoding the components of the inducible branched-chain ketoacid dehyd rogenase, BkdR, a positive regulator of the bkd operon and a homolog o f Lrp of Escherichia coli is encoded by a structural gene adjacent to, and divergently transcribed from, the bkd operon of P, putida. BkdR w as purified from E. coli containing bkdR cloned into pCYTEXP1, an expr ession vector, The molecular weight of BkdR obtained by gel filtration indicates that BkdR is a tetramer, and the abundance of BkdR in P, pu tida was estimated to be about 25 to 40 copies of the tetramer per cel l, BkdR bound specifically to the region between bkdR and bkdA1, the l atter being the first gene of the bkd operon, One BkdR-DNA complex,vas observed in gel mobility shift patterns, Approximately 100 bp was pro tected from the action of DNase I by BkdR, and the addition of L-branc hed-chain amino acids enhanced the appearance of hypersensitive sites in the protected region, There are four potential BkdR DNA binding seq uences in this region based on similarity to Lrp-binding consensus seq uences, Like many other transcriptional activators, BkdR regulates exp ression of its structural gene, DNAs from several gram-negative bacter ia hybridized to a probe containing bkdR, indicating the presence of b kdR-like genes in these organisms,